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10348 • The Journal of Neuroscience, June 19, 2013 • 33(25):10348 –10361
Development/Plasticity/Repair
Kinetic Analysis of npBAF to nBAF Switching Reveals
Exchange of SS18 with CREST and Integration with Neural
Developmental Pathways
Brett T. Staahl,1 Jiong Tang,1 Wei Wu,1 Alfred Sun,1 Aaron D. Gitler,2 Andrew S. Yoo,3
and Gerald R. Crabtree1
1
3
Departments of Developmental Biology and Pathology, and 2Department of Genetics, Stanford University Medical School, Stanford, California 94305, and
Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110
During the development of the vertebrate nervous system, neural progenitors divide, generate progeny that exit mitosis, and then migrate
to sites where they elaborate specific morphologies and synaptic connections. Mitotic exit in neurons is accompanied by an essential
switch in ATP-dependent chromatin regulatory complexes from the neural progenitor Brg/Brm-associated factor (npBAF) to neuronspecific nBAF complexes that is in part driven by miR-9/9* and miR-124. Recapitulating this microRNA/chromatin switch in fibroblasts
leads to their direct conversion to neurons. We have defined the kinetics of neuron-specific BAF complex assembly in the formation of
induced neurons from mouse embryonic stem cells, human fibroblasts, and normal mouse neural differentiation and, using proteomic
analysis, found that this switch also includes the removal of SS18 and its replacement by CREST at mitotic exit. We found that switching
of chromatin remodeling mechanisms is highly correlated with a broad switch in the use of neurogenic transcription factors. Knock-down
of SS18 in neural stem cells causes cell-cycle exit and failure to self-renew, whereas continued expression of SS18 in neurons blocks
dendritic outgrowth, underlining the importance of subunit switching. Because dominant mutations in BAF subunits underlie widely
different human neurologic diseases arising in different neuronal types, our studies suggest that the characteristics of these diseases must
be interpreted in the context of the different BAF assemblies in neurons rather than a singular mammalian SWItch/sucrose nonfermentable (mSWI/SNF) complex.
Introduction
Combinatorics frequently underlie biologic specificity. Examples
include the production of proteins from the combinatorial assembly of 20 aa and perhaps most remarkably the connectivity of
the nervous system. Specific patterns of chromatin regulation can
also be produced by combinatorial assembly of subunits of chromatin regulatory complexes. The first examples of this were the
family of mammalian SWItch/sucrose nonfermentable (mSWI/
SNF) or Brg/Brm-associated factor (BAF) complexes (Wang et
al., 1996a; Wang et al., 1996b; Lemon et al., 2001; Lessard et al.,
2007), which are made up of ⬃15 subunits now known to be
Received March 21, 2013; revised May 8, 2013; accepted May 15, 2013.
Author contributions: B.T.S., W.W., A.S.Y., and G.R.C. designed research; B.T.S., J.T., W.W., and A.S. performed
research; A.D.G. contributed unpublished reagents/analytic tools; B.T.S., J.T., A.D.G., A.S.Y., and G.R.C. analyzed
data; B.T.S. and G.R.C. wrote the paper.
This work was supported by grants from the Howard Hughes Medical Institute, the National Institutes of Health
(Grant #CA163915 and Grant #NS046789), and the California Institute of Regenerative Medicine (Grant #CIRM
R34-05886 to G.R.C.). B.T.S. was supported by the Developmental and Neonatal Biology Training Program (Grant
#T32 HD007249 from the National Institutes of Health). A.X.S. was supported by the Agency of Science, Technology
and Research of Singapore (A*STAR). A.S.Y. was a fellow of Helen Hay Whitney Foundation. We thank all members
of the Crabtree Laboratory for engaging discussions on the subject, especially Julie Lessard, Jiang Wu, and Ary Shalizi
for helpful suggestions.
Correspondence should be addressed to Gerald R. Crabtree, Investigator, Howard Hughes Medical Institute, Rm
B211, Beckman Center HHMI, 279 Campus Drive, Stanford University School of Medicine, Stanford, CA 94305-5323.
E-mail: [email protected].
DOI:10.1523/JNEUROSCI.1258-13.2013
Copyright © 2013 the authors 0270-6474/13/3310348-14$15.00/0
encoded by 28 genes (Lessard et al., 2007; Wu et al., 2009) and this
study). These complexes are assembled from homologous subunits encoded by gene families in much the same way that letters
are assembled into words to produce specific meanings. For specific complexes to have specific biological meanings, the subunits
must not be readily exchanged, because this would produce a
reduction in meaning in the same way that indiscriminately
switching the “W” in the word “NOW” for “T” would produce
confusion. Accordingly, BAF complexes appear to be assembled
with nonexchangeable, dedicated subunits (Zhao et al., 1998).
Perhaps the most distinctive and specific of these chromatin regulatory complexes is found in the mammalian nervous system,
where neuron-specific BAF (nBAF) complexes are distinguished
by nonexchangeable subunits apparently dedicated to these complexes: BAF53b, BAF45b, and BAF45c (Lessard et al., 2007).
Recent evidence suggests that the subunit composition of BAF
complexes might have an instructive developmental role. In the
nervous system, the transition from the neural progenitor BAF
(npBAF) complexes in neural progenitors to nBAF complexes in
neurons is controlled by two microRNAs, miR-9/9* and miR124. These microRNAs bind to the 3⬘ untranslated region of
BAF53a, repressing its expression and making way for substitution with the neuron-specific BAF53b (Yoo et al., 2009). Recapitulation of this microRNA/chromatin switch in human fibroblasts
results in their conversion to functional neurons with as few as
just one neurogenic factor, NeuroD2 (Yoo et al., 2011).
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
The possible instructive role of nBAF complexes is further
supported by studies of human neurologic diseases. Recently, de
novo mutations in BAF subunits have been identified in a number
of neurological diseases. Five BAF subunits, BRG, BAF250a
(ARID1a), BAF250b (ARID1b), BAF57, and BAF47, were linked
to Coffin-Siris Syndrome (CSS), a sporadic intellectual disability
syndrome (Santen et al., 2012; Tsurusaki et al., 2012). Eightyseven percent of CSS patients in these studies had a mutation in a
BAF subunit, underscoring how mutations in genes from the
same protein complex can have similar clinical presentations. In
addition, BRM, one of the two ATPases of BAF complexes, was
found to be mutated in 36 of 44 (82%) cases of NicolaidesBaraitser syndrome (NBS), which is characterized by mild to severe mental retardation, specific language difficulties, and
microcephaly (Van Houdt et al., 2012). In addition, mutations in
BAF250b are common in sporadic mental retardation (Halgren et
al., 2012; Hoyer et al., 2012). Population genetic studies have also
implicated BRM and BAF250b in schizophrenia (Koga et al.,
2009; Loe-Mie et al., 2010) and exome sequencing of autistic
individuals revealed mutations in BAF155, BAF170, BAF180,
BAF250b, and REST (Neale et al., 2012; O’Roak et al., 2012), the
latter of which controls miR-9/9* and miR-124 (Conaco et al.,
2006). All of these mutations are dominant, perhaps paralleling
the dominant role of Brg and BAF155 in neural development
(Bultman et al., 2000; Kim et al., 2001).
nBAF chromatin-remodeling complexes have an evolutionary conserved role in dendrite morphogenesis. Drosophila
BAP55, BAP60, Snr1, and Brm, the fly homologs of BAF53b,
BAF60c, BAF47, and BRM, respectively, are required for routing
of dendrites in the peripheral nervous system (Parrish et al.,
2006). Furthermore, mutation of BAP55 causes perfect retargeting of CNS olfactory projection neuron dendrites from one
glomerulus to another (Tea and Luo, 2011), a phenotype that was
rescued by human BAF53b. BAF53b-null mice have impaired
activity-dependent dendritic outgrowth (Wu et al., 2007). These
studies suggest that failures of dendritic morphogenesis or targeting could underlie human neurologic disorders characterized by
mutation of nBAF subunits.
To understand the mechanistic details of BAF subunit switching during neuronal differentiation, we undertook a proteomic
and kinetic analysis of BAF complexes as neural progenitors become neurons. We show that SS18 is replaced by its paralog,
CREST, near mitotic exit. We have related the switching of subunits to the expression patterns of classic neurogenic and neuronal genes to establish the characteristics of the genetic circuitry
used during both differentiation of embryonic stem (ES) cells to
neurons and direct reprogramming of human fibroblasts into
neurons.
Materials and Methods
Affinity purification and mass spectrometry. Affinity purification and mass
spectrometry of endogenous BAF complexes were performed as described previously (Lessard et al., 2007; Ho et al., 2009b).
Preparation of ES cell and brain nuclear extracts. Mouse CD1 embryonic brains of either sex were dissected in cold HBSS, transferred to cold
hypotonic lysis buffer containing the following: 25 mM HEPES, pH 7.6, 5
mM MgCl2, 25 mM KCl, 0.05 mM EDTA, 10% glycerol, 0.1% NP40, 1 mM
DTT, and protease inhibitors (complete mini tablets; Roche) supplemented with 1 mM PMSF, and homogenized with 10 –15 strokes of a glass
Dounce homogenizer. Nuclei were centrifuged at 800 ⫻ g for 3 min.
Chromatin was precipitated in 10 mM HEPES, pH 7.6, 3 mM MgCl2, 100
mM KCl, 0.1 mM EDTA, 10% glycerol with 1:10 vol of 3 M ammonium
sulfate, pH 7.9, for 30 min and centrifuged in an Ultracentrifuge at
100,000 rpm for 15 min. Proteins were then precipitated with 0.3 g/ml
J. Neurosci., June 19, 2013 • 33(25):10348 –10361 • 10349
high-grade ammonium sulfate for 20 min and spun at 100,000 rpm for 15
min. Protein extracts were resuspended in immunoprecipitation buffer
containing the following: 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 1% Triton X-100, and 1 mM DTT with protease inhibitors unless
stated otherwise.
Glycerol gradient cosedimentation experiments. For glycerol gradient
sedimentation, embryonic day 10.5 (E10.5) or E18.5 brain nuclear extracts were diluted in 37.5 mM HEPES-KOH, pH 7.9, 90 mM KCl, 6.25
mM MgCl2, 0.05 mM EDTA, and 0.75 mM DTT. Approximately 500 ␮g
(in 500 ␮l) of nuclear extract was applied to 10 ml 10 –30% column
gradients of glycerol in HEMG/100 buffer containing 100 mM KCl. Samples were centrifuged in a Beckman SW40Ti rotor at 32,000 rpm for 20 h
at 4°C. Nineteen 500 ␮l fractions were collected from the top (no. 1) to
the bottom (no. 19) of the gradients by hand. Fractions were separated by
SDS-PAGE and analyzed by immunoblotting with anti-Brg, BAF250a,
BAF170, BAF155, CREST, SS18, BAF53a, 53b, and Ezh2 specific
antibodies.
Immunocytochemistry. Cells were grown on glass coverslips coated
with poly-ornithine, laminin, and fibronectin. Cells were fixed with 4%
PFA, blocked, and permeabilized with 3% normal goat serum,5% BSA,
0.1% Triton X-100, and incubated with primary antibodies overnight,
followed by goat anti-rabbit or mouse secondary antibodies (Invitrogen)
and DAPI, washed with PBS, and mounted with 4% N-propyl gallate
(Sigma) in 90% glycerol, 10% PBS and imaged on a Leica DM5000 fluorescent microscope.
Co-IP experiments. Nuclear extracts were made as described above.
Co-IP experiments were performed with 100 –200 ␮g nuclear extracts
precleared with either protein A or G beads (Sepharose or Dynabeads),
incubated with primary antibodies at 4°C overnight, bound to protein A
or G beads, washed 3 times, and interacting proteins eluted with 2⫻
sample buffer and boiling (Fig. 2, Fig. 8) or 0.1 M glycine, pH 2.5 (Fig. 3),
before SDS-PAGE and Western analysis.
Antibodies. mouse anti-b-III tubulin (Tuj) (Covance, Western [W],1:
30k; immunofluorescence [IF],1:20k), rabbit anti-BAF170 (39A Bethyl
Laboratories, rabbit anti-BAF155 (-5 or -3-glycerol gradients; Crabtree
Laboratory; W, 1:5k), mouse anti-Brg1-G7 (Santa Cruz Biotechnology;
W, 1:2k), rabbit anti-SS18 (H80; Santa Cruz Biotechnology; W, 1:750; IF,
1:300), goat anti-CREST (M15; Santa Cruz Biotechnology; W, 1:1000; IF,
1:3k), CREST (Proteintech; IF, 1:2k), rabbit anti-BAF250a (H-90x
SC98441; Santa Cruz Biotechnology; W, 1:3k), mouse anti-BAF250a
(Santa Cruz Biotechnology; W, 1:1k), mouse anti-BAF250b (SC32762;
Santa Cruz Biotechnology; W, 1:6k), mouse anti-BRM (Santa Cruz Biotechnology; W, 1:2k), rabbit anti-BAF53a (Novus; W, 1:5k; IF, 1:2k),
rabbit anti-53b (fraction 2–3, BAF53a depleted; Crabtree laboratory; W,
1:250; IF, 1:250), GFAP (MAB3402; Millipore; IF, 1:1k), nestin (BD Biosciences; IF, 1:2k), mouse anti-V5 (Invitrogen), DAPI, and Alexa Fluor
488, Alexa Fluor 568 (Invitrogen) secondary antibodies (IF, 1:1k). IRDye
secondary antibodies included: goat anti-Mouse 800CW (926 –32210;
LiCOR), goat anti-rabbit 800CW (926 –32211; LiCOR, ), goat antimouse 680 (926 –32220; LiCOR), and goat ani-rabbit 680 (926 –32221;
LiCOR, ; W, 1:30k). Western blots were imaged using the LiCOR Odyssey infrared imager.
Cell conversion. The conversion of mouse ES cells into glutamatergic
pyramidal neurons followed essentially the protocol in Bibel et al. (2007).
Conversion of human fibroblasts into neurons followed the protocol in
Yoo et al. (2011).
Quantitative RT-PCR. For quantitative RT-PCR (qRT-PCR), total
RNA was isolated using TRIzol reagent (Invitrogen). One microgram of
total RNA was treated with DNase I to remove potential contamination
of genomic DNA. One microgram of DNase I-treated RNA was reverse
transcribed using a First Strand Synthesis kit (Invitrogen) and ultimately
resuspended in 900 ␮l of water. qRT-PCR analysis was performed in
duplicate using 1/100 of the reverse transcription reaction in an ABI
Prism 7000 (Applied Biosystems) with FastStart Universal SYBR green
Master Mix with ROX (Roche). Data were extracted from the linear
range of amplification. All graphs of qRT-PCR data shown represent
samples of RNA that were DNase treated, reverse transcribed, and amplified in parallel to avoid variation inherent in these procedures. The
housekeeping gene Ppia was used for sample cDNA normalization be-
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
10350 • J. Neurosci., June 19, 2013 • 33(25):10348 –10361
A
Protein
CELL TYPE
MEF
P0 Brain
ESC
%cov #pept
B
10%
1
2
3
4
5
6
7
8
30%
glycerol
9 10 11 12 13 14 15 16 17 18 19
BAF250a
%cov #pept %cov #pept
BAF250b
total (unique)
45.7 243(101) 34.3
166(68) 25.4
208(46)
BAF155 (Smarcc1)
62.8 377(130) 54.4
207(80) 19.3
43(15)
SS18 (SYT)
39.3
28(14)
BAF53a (Actl6a)
52.9
62(26) 37.1
BAF45a (PHF10)
20.8
7(7) 21.3
BAF250a (Arid1a)
55.5
BAF250b (Arid1b)
17.5
Brm (Smarca2)
N.D.
3.7
7(3)
0
58(18) 29.1
7(7)
0
49(14)
14.5
13(6)
90(34) 40.8
40(21) 27.7
278(86)
31(27) 45.3
72(52) 16.4
95(34)
1(1)
46.9 116(48) 20.4
63(22)
0
5.8
1(1) 23.3
15(8)
20(14) 19.5
69(15)
BAF180/pBrm
E10.5 cortex
Brg (Smarca4)
Brg
BAF155
BAF170
SS18
BAF53a
CREST
BAF53b
Ezh2
BAF250a
0
BAF60c (Smarcd3)
19.3
5(3)
30.8
CREST (SS18l1)
6.1
3(3)*
2.9
2(2)*
3.2
16 (3)
BAF53b (Actl6b)
0
0
0
0
33.6
59(17)
BAF45b (DPF1)
23.2
8.1
2(2)
52.7
87(23)
BAF45c (DPF3)
0
0
5.8
1(1)
23.3
15(8)
54
35(27)
27
20(14) 24.9
75(18)
0
0
0
10(7)*
BAF250b
BAF180/pBrm
Brm
Brg
E18.5 cortex
BAF170 (Smarcc2)
BAF155
BAF170
SS18
BAF53a
CREST
BAF53b
BAF47 (Smarcb1)
Ezh2/Bmi
0
0
0
Ezh2
<100kDa
~2MDa
>5mDa
Figure 1. Identification of novel BAF subunits SS18 and CREST. A, Table of mass-spectrometric analysis of proteins co-purified with Brg/Brm from mouse ES cells, MEFs, and newborn P0 whole
brain. Shown is the percent coverage based on unique peptides. B, Glycerol gradient (10 –30%) analysis of E10.5 and E18.5 mouse brain cortical nuclear extracts. SS18 but not CREST cosediments
with npBAF complexes at 2MDa in E10.5 mouse brain nuclear extracts, whereas CREST cosediments with nBAF complexes in E18.5 mouse brain nuclear extracts. The Polycomb PRC2 complex protein
Ezh2 was blotted as a control and peaks in ⬃1 MDa fractions. *From other studies, these proteins are not found in pure undifferentiated ES cells nor MEFs. The presence of these proteins in ES cells
could be from spontaneously differentiating cells in the ES cultures and neural cells in the MEFs. Glycerol gradients are representative of two separate protein purifications from each developmental
time point.
cause it changed the least across all data points of the time course. Error
bars represent SEM of three biological replicates.
Primary neuron cell culture. mouse E18.5 cortical neurons of either sex
were isolated using the MACSTM neuron isolation kit. Dissociated cells
were transfected with GFP or other constructs using Nucleofector
(Amaxa shuttle) before plating on poly-L-ornithine, laminin-, and
fibronectin-coated coverslips. Culture media contained DMEM/F12
with putrescine, 2-mercaptoethanol, transferrin, insulin, selenium, progesterone, MEM vitamin additive, and 5% FBS.
Dendrite outgrowth experiments. Mus musculus SS18, Ascension number BC096742 and Mus musculus CREST ascension number BC053087
cDNA were ordered from OpenBioSystems and subcloned into the
pCIG-IRES-eGFP plasmid. Then, 25 ng of the respective plasmids mixed
with 475 ng pCIG-IRES-eGFP plasmid was transfected into 250,000 dissociated E18.5 mouse cortical neurons of either sex using Nucleofector
(AMAXA shuttle) and plated onto coated coverslips. The neural cultures
were grown for 6 d with or without 30 mM KCl for the last day. To define
dendrite structure, cultures were fixed with 4% PFA and stained with
anti-GFP (Invitrogen), anti-MAP2 (Sigma), and Alexa Fluor 488 and
Alexa Fluor 568 (Invitrogen) secondary antibodies (1:1000). Dendrite
analysis was done on pictures of GFP ⫹/MAP2 ⫹ neurons using ImageJ
and NeuronJ software.
Results
SS18 and CREST are mutually exclusive npBAF and
nBAF subunits
An unbiased proteomic comparison was performed with murine
ES cells, mouse embryonic fibroblasts (MEFs) and newborn (P0)
brain extracts. BAF complexes were affinity purified using a highaffinity, highly specific Brg/Brm antibody (J1) that recognizes an
epitope without complex disruption (Khavari et al., 1993). Brg/
Brm-associated proteins were subjected to proteolytic digestion,
analyzed by multidimensional liquid chromatography electrospray ionization-tandem mass spectrometry, and identified using
SEQUEST (Yates et al., 1995) to determine the identity and relative abundance of each protein in the BAF complexes (Ranish et
al., 2003).
This analysis identified the paralogs SS18 (aka SYT) and
CREST (SS18L1) (62% identical) as Brg/Brm-interacting proteins, which is in agreement with previous reports (Wu et al.,
2007; Qiu and Ghosh, 2008; Middeljans et al., 2012). We identified unique SS18 peptides for ES cells (28 peptides, 39.3% coverage), MEFs (7 peptides, 13.7% coverage), but not for P0 brain
nuclear extracts. In contrast, CREST peptides were most abundant in P0 brain (Fig. 1A), consistent with the expression pattern
of CREST described previously (Aizawa et al., 2004). The reciprocal pattern of the recovered peptide abundance also suggested a
switch in SS18/CREST expression during neural differentiation,
in which SS18 is expressed in ES cells and MEFs whereas CREST
is expressed in neurons.
Because specific assemblies of subunits provide the biochemical underpinnings for biologic specificity, we determined
whether SS18 and CREST were dedicated BAF subunits by examining their sedimentation on glycerol gradients (Fig. 1B). This
analysis is useful for differentiating between interacting proteins
that might exchange quickly and subunits that are structurally
part of a protein complex. This is an important functional point
because it defines the potential for new biologic meaning to
emerge from different complex assemblies. In E10.5 cortical
preparations (composed of ⬎95% Pax-6 ⫹, BAF53a ⫹ neural progenitors; data not shown), SS18 sedimentation is in fractions 15
and 16 with 2 MegaDalton markers. Brg/Brm, the core ATPases
of these BAF complexes, and the subunits BAF250a (Arid1a),
BAF250b (Arid1b), BAF170, BAF155, and BAF53a also cosediment in these fractions. There was no SS18 in the earlier fractions
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
J. Neurosci., June 19, 2013 • 33(25):10348 –10361 • 10351
Figure 2. SS18 and CREST are mutually exclusive, nonexchangeable subunits of npBAF and nBAF complexes, respectively. A, Co-IP from E15.5 brain cortical extracts; Brg co-IPs SS18 and CREST
whereas CREST does not co-IP SS18 and SS18 does not co-IP CREST. Black arrowheads mark the SS18 and CREST proteins. Empty arrowhead marks SS18 Ab cross-reacting with CREST (CREST runs
above SS18 on these Western blots). *Immunoglobin heavy chain. B, Immunostaining on cross sections of E12.5 spinal cords. SS18 is expressed specifically in neural progenitors in ventricular zone
(VZ) and is replaced by the homologous subunit CREST in postmitotic neurons in the postmitotic zone (PMZ). Scale bar, 250 ␮m. C, Mutual exclusive expression pattern of SS18 and CREST as
determined by costaining primary mouse E15.5 cortical cells cultured for 4 d in vitro with neural markers. SS18 is expressed in Nestin ⫹ NS/progenitors, whereas CREST is specifically expressed in
Tuj ⫹ neurons. Scale bar, 20 ␮m. D, Quantification of staining in C. Data are ⫾ SD of three fields of view each, with ⬃100 cells each field.
in which monomeric proteins would sediment. Polybromo-1
(PBrm/BAF180) sediments primarily in a distinct higher molecular weight complex termed PBAF. CREST was not present in
E10.5 cortical preparations nor was BAF53b. In contrast, E18.5
cortical preparations composed of 75% postmitotic, BAF53b ⫹
neurons (data not shown), CREST, and BAF53b cosedimented
exclusively with Brg. The appearance of low levels of SS18 in the
E18.5 gradients was due to neural progenitors and glia present at
E18.5. Therefore, during the course of neuronal development,
CREST is activated and incorporated into nBAF complexes.
These studies indicate that SS18 and CREST are dedicated to the
2 MDa Brg/Brm-based BAF complexes.
One could imagine that SS18 and CREST can heterodimerize
and are both present within a single complex or that there is a
sterically specific concavity within BAF complexes that is occupied by either SS18 or CREST— but never both. To test these
possibilities, we conducted co-IP experiments with antibodies
against SS18, CREST, or Brg in mouse E15.5 cortical nuclear
extracts. Because the E15.5 mouse cortex contains a mixture of
neural progenitors and neurons, this experiment tests both the
mutual exclusivity of the subunits and the exchangeability of
subunits. Antibodies to SS18, CREST, and Brg co-IP the core BAF
subunits Brg, BAF155, and BAF170. Only antibodies to Brg co-IP
both SS18 and CREST, whereas antibodies to SS18 co-IP SS18 but
not CREST and antibodies to CREST co-IP CREST but not SS18
(Fig. 2A). This failure to co-IP homologous subunits indicates
that SS18 and CREST are mutually exclusive BAF subunits, with
few if any complexes having both SS18 and CREST. These data
also suggest the subunits are not easily exchangeable between
complexes in vitro.
To determine whether the mutually exclusive assembly was
due to distinct patterns of expression, we determined the cellular
localization and the cell-type specificity of SS18 and CREST. In
the developing spinal cord between E10.5 and E16.5, SS18 was
specifically expressed in the proliferating neural progenitors of
the ventricular zone. In differentiated neurons of the postmitotic
zone, SS18 was repressed and CREST was expressed (Fig. 2B). In
primary isolated cortical neural cultures, SS18 was present in
⬎75% of Nestin ⫹ neural progenitors and ⬍5% of ␤-tubulin III ⫹
(Tuj ⫹) neurons. Conversely, CREST was exclusively dedicated to
Tuj ⫹ neurons (Fig. 2C,D). Therefore, we conclude that SS18 is an
es/npBAF subunit and CREST is an nBAF subunit, and different
cell types are characterized by specific BAF complexes that have
the potential for unique biologic functions.
SS18 and CREST are subunits of the npBAF and nBAF
complexes, respectively
To define the stability of SS18 and CREST incorporation into
npBAF and nBAF complexes, respectively, we determined their
resistance to increasing concentrations of the denaturant urea.
Urea will denature most proteins at ⬃5 M and will partially denature many proteins at lower concentrations. Therefore, the
sensitivity of a protein–protein interaction to urea is a measure of
the degree to which the interaction depends on the native state of
the protein. Interactions that survive high concentrations of urea
are often found to reflect cofolding within complexes such as the
ribosome (Petermann and Pavlovec, 1971) and are useful in differentiating a subunit from an associated, exchangeable protein.
BAF complex core subunits remain associated with Brg ⬎1 M
urea (core subunits also remain associated under highly stringent
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
10352 • J. Neurosci., June 19, 2013 • 33(25):10348 –10361
IP conditions of 0.3 M NaCl, 1% NP40,
0.5% deoxycholate, and 0.1% SDS; Wang
et al., 1996a; Zhao et al., 1998; Lessard et
al., 2007). We treated ES cells (Fig. 3A),
E10.5 neural progenitors, E18.5 neurons
(Fig. 3B), and P1.5 cortical cell (Fig. 3C)
nuclear extracts with increasing amounts
of urea for 10 min at room temperature
before IP with an antibody to Brg. The
bona fide core BAF subunits BAF155 and
BAF170 remained associated with Brg up
to 2.5 M urea, whereas SS18 and CREST
interactions survived up to 5 M urea, indicating extensive and stable protein–protein interactions that are unlikely to turn
over quickly supporting the SS18 and
CREST co-IP results.
These studies indicate that the two
subunits are likely to have interlocking interactions dependent upon their native
folded state within BAF complexes and
are not dependent upon BAF155 or
BAF170 to be bound to the complexes.
They also indicate that the SS18 and
CREST subunits within BAF complexes
have approximately the same stability as
the subunits of ribosomes.
A
Embryonic Stem Cells
Brg IP
NE
IgG
-
.25
1
2.5
5 M Urea
Brg
BAF155
BAF53a
SS18
TBP
B
E10.5 Neural Progenitors
Brg IP
NE IgG -
1 2.5 5M
E18.5 Neurons
Brg IP
NE IgG -
1 2.5 5M Urea
Brg
BAF250b
BAF180/pBrm
BAF155
BAF170
SS18
CREST
Ex vivo production of neurons
TBP
also involves substitution of
SS18-containing npBAF complexes for
P1.5 Neonatal Cortex
C
CREST-containing nBAF complexes
Brg IP
To further define the coregulation of
SS18/CREST with other npBAF and nBAF
- 1 2.5 5 M Urea
NE
IgG
subunits during the course of neural difBrg
ferentiation, we set up an ex vivo neural
BAF170
differentiation system that converts ES
cells into glutamatergic neurons (Bibel et
CREST
al., 2007). In this ES-to-neuron system,
⫹
⫹
cells differentiate into Nestin /Pax6 ,
TBP
radial glial cell-like neural progenitor
cells, when treated with retinoic acid (RA; Figure 3. SS18 and CREST remain associated with Brg up to 5 M urea. A, Denaturation analysis of BAF complexes in ES cells. SS18
Fig. 4A). As they did in vivo, these neural and BAF53a remain associated with Brg at 5 M urea, whereas BAF155 comes off in 2.5 M urea. Nuclear extracts were prepared from
progenitors differentiated into neurons E14 ES cells as described in the text. Nuclear extracts were subjected to different concentrations of urea for 10 min at 25°C before IP,
that form functional synaptic contacts. diluted 10⫻ with IP buffer, incubated with anti-Brg (G7) antibodies overnight at 4C. Brg co-IP proteins were isolated, electrophoWe used this system to visualize the kinet- resed, and probed with antibodies to the labeled proteins. B, Denaturation analysis of npBAF complexes in E10.5 neural progeniics of npBAF repression and nBAF activa- tors or E18.5 neurons. npBAF containing SS18 in E10.5 nuclear extracts switches to nBAF containing CREST in E18.5 neurons. SS18
tion with antibodies to BAF subunits, the remains associated with Brg to 5 M urea in neural progenitors, whereas CREST remains associated with Brg to 5 M urea in neurons.
neural progenitor marker Nestin, or the No CREST is detected at E10.5 and very little SS18 is detected at E18.5. TBP is blotted as a loading control. C, Denaturation analysis
of nBAF complexes in P1.5 brain extracts. CREST remains associated with Brg up to 5 M urea, whereas BAF170 comes off at 2.5 M
neuron marker Tuj (Fig. 4B). We deter- urea. Western blots are representative of three separate protein purifications from the respective cell types.
mined the timing of mitotic exit by
5-ethynyl-2⬘-deoxyuridine (EdU) pulsing
neurons, SS18 transcription was reduced to 25% of maximum at
to be between day 9 and day 10 (Fig. 4C). Interestingly, this time
day 10 and protein levels decreased significantly on day 11 (Fig.
point corresponds to the switch from npBAF subunits to nBAF sub5 A, B). In this neuronal differentiation system, on day 12 among
units. At this time, expression of npBAF subunits (SS18, BAF45a,
viable cells, 80 ⫾ 3% were Tuj ⫹ neurons, ⬍1% were Nestin ⫹
BAF53a) was repressed as the nBAF subunits (CREST, BAF45b/c,
neural progenitors, and 19 ⫾ 3% were non-neural lineage cells.
BAF53b) were activated. However, we noted a low level of expresThe non-neural cells were SS18 ⫹ (data not shown). Therefore,
sion of BAF45c in neural progenitors, the appearance of which was
the residual SS18 protein detected by the Western blots was from
earlier than that of CREST, BAF45b, or BAF53b.
non-neural cells in the culture. In a reciprocal fashion to SS18,
To further examine the nature of the switching mechanism,
CREST was activated to 80% of maximum by day 10, with robust
we measured mRNA and protein levels during the switch. The
protein accumulation observed as the cells differentiated into
npBAF to nBAF switch is rapid as nBAF subunits are robustly
neurons on day 10 (Fig. 5 A, B). Because the SS18/CREST switch
activated and npBAF subunits are repressed. In these induced
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
J. Neurosci., June 19, 2013 • 33(25):10348 –10361 • 10353
is similar to the BAF53a/53b switch, we
wondered how this ES-to-neuron differentiation system recapitulated the essential genetic circuitry we defined previously
for the miRNA/chromatin switch during
neural development. At the top of this circuit, REST, which with its co-repressors
restricts the activation of neuronal genes
to neurons by silencing its target genes in
non-neurons, is downregulated as neural
progenitors differentiate into neurons
(Chong et al., 1995; Schoenherr and Anderson, 1995; Chen et al., 1998; Lunyak et
al., 2002; Kuwabara et al., 2004; Ballas et
al., 2005). The REST target genes miR9/9* and miR-124 are derepressed, which
leads to repression of BAF53a and substitution with BAF53b during neuronal differentiation (Conaco et al., 2006; Yoo et
al., 2009).
In the ex vivo differentiation system,
the neural progenitor genes Nestin and
Pax6 were activated as cells passed
through the neural progenitor stage. The
proneural genes Neurogenin2 (Ngn2)
and Ascl1 preceded the activation of the
neural differentiation genes NeuroD2 and
Myt1L, recapitulating their in vivo order
of activation (Bertrand et al., 2002). REST
levels were reduced at the mRNA and protein level 4 d after LIF removal. On day 6,
as cells entered the Nestin ⫹/Pax6 ⫹ neural
progenitor phase 2 d after the addition of
RA, REST mRNA was reduced to 25% of
ES cell levels with marked reduction in
REST protein. On day 7, the REST target
genes miR-9/9* were derepressed and
activated and, on day 8, miR-124 was
activated. Therefore miR-124 activation
lagged approximately 1 d behind miR-9/
9*. The additional REST target genes
BAF53b and BAF60c (Johnson et al.,
2008) were activated on day 8 and 9,
respectively, when REST transcript levels dropped ⬍10% of maximum (Fig.
5 A, B).
4
Figure 4. Kinetics of induction and loss of nBAF and npBAF subunits after neural induction of ES cells. A, Schematic of neural
induction of ex vivo ES cell-to-neuron differentiation time course. Open arrowhead denotes cell aggregate dissociation and transfer
of neural progenitors to neural differentiation media and polyornithine-, laminin-, and fibronectin-coated plates. B, SS18
protein intensity decreases when cells differentiate into Tuj ⫹
neurons. CREST is first detected on day 9 in Tuj ⫹ neurons with
rapidly increasing intensity as the cells become more mature
neurons. BAF53a/45a decrease in neurons and BAF53b/45b
are activated only in Tuj ⫹ neurons. BAF53a colocalizes with
Nestin ⫹ NS/progenitor cells. BAF45c is activated before
BAF45b and before cells becoming Tuj ⫹. C, Proliferation measured by EdU incorporation during the switch from Nestin ⫹
NS/progenitors to Tuj ⫹ neurons. Cell cycle exit corresponds
with the timing of the npBAF to nBAF switch. Cells were pulsed
with EdU for 2 h before being fixed. Data are shown ⫾ SEM.
Images and EdU staining are representative of three separate
ex vivo differentiations.
10354 • J. Neurosci., June 19, 2013 • 33(25):10348 –10361
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
Figure 5. Kinetics of transcriptional induction and repression of nBAF and npBAF genes, miRNAs, cell-type marker genes, and neurogenic transcription factors after neural induction of ES cells.
A, qRT-PCR analysis of mRNA and microRNAs during neural induction time course. Green panel denotes neural progenitor stage, red panel denotes neuron stage, and vertical line delineates time of
cell cycle exit. B, Protein analysis by Western blot. BAF45c is the first nBAF subunit to be activated and BAF53b is the last to be activated. Data are representative of three separate ex vivo
differentiations. C, BAF45c is activated independent of RA induction. BAF45b and BAF53b are not activated without RA induction. D, BAF250a, 250b, BAF60a, BAF60b, and BAF60c expression kinetics.
qRT-PCR is presented as a percentage of the maximum transcript level ⫾ SEM of three separate ex vivo differentiations.
BAF53a repression was observed on day 10 as the miRNAs
rapidly approached their maximal levels, reflecting the essential
role of miR-9/9* and miR-124 for BAF53a repression. The
miRNAs were robustly activated: miR-9/9* was upregulated
⬃1000-fold and miR-124 was upregulated ⬃100-fold. The reduction of BAF53a protein lagged behind its transcript kinetics,
possibly because of the stability of this protein and the presence in
non-neural cells in the culture. Interestingly, BAF45c was induced before miR-9/9* and miR-124 and was activated in the
absence of RA, albeit not as robustly (Fig. 5C), indicating that the
activation of BAF45c must be independent of RA induction and
the expression of miR-9/9* and miR-124. miR-9/9*, miR-124,
CREST, BAF45b, and BAF53b were not activated in the absence
of RA induction (Fig. 5C and data not shown). Therefore, there
are at least two upstream operators that bring about the npBAF to
nBAF switch, one RA dependent and one RA independent. The
switch to nBAF subunits occurs on day 10, as measured by transcript and protein (Fig. 5) corresponding to immunostaining and
cell cycle exit data (Fig. 4). We conclude the ex vivo ES-to-neuron
differentiation system recapitulates the sequence and timing of
the in vivo microRNA/chromatin switch.
The dynamic regulation during ex vivo differentiation was
determined for the BAF subunits BAF250a (Arid1a) and
BAF250b (Arid1b), which have been genetically linked to mental
retardation and autism (see Discussion). BAF250a was more
abundant than BAF250b in ES cells. However, BAF250b mRNA
was induced 25-fold in neurons relative to ES cells (Fig. 5D).
BAF250b protein levels were 2⫻ higher in E18.5 neurons relative
to E10.5 neural progenitors (Fig. 3B, Western blot densitometry
normalized to TBP levels). Therefore, BAF250b was transcriptionally activated during neuronal differentiation. In addition,
BAF250b appeared to be regulated at the level of pre-mRNA
splice variation as the relative intensities of the BAF250b bands
changed from E10.5 neural progenitors to E18.5 neurons (Fig. 1B,
Fig. 3B). BAF250b has 4 predicted splice variants (http://www.
ensembl.org). Interestingly, the lowest-molecular-weight band was
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
Figure 6. SS18 and CREST switch during artificial neuron development. Human fibroblasts
were infected with lentivirus containing miR-9/9* and miR-124. Eighteen days later, mRNA was
isolated. qRT-PCR analysis shows the switch from SS18-containing BAF complexes in fibroblasts
to predominantly CREST-containing complexes after miR-9/9* and miR-124 addition to human
fibroblasts. After the switch, the transcript profile is similar to that of an adult human brain sample.
Data are shown ⫾ SEM of three separate neuronal inductions. *p ⬍ 0.05, Student’s t test.
most tightly associated with BAF complexes because it remained
associated with Brg in 5 M urea (Fig. 3B).
The ex vivo differentiation system also recapitulates previously
observed expression patterns for BAF60a, BAF60b, and BAF60c.
BAF60a and BAF60b were expressed in ES cells whereas BAF60c
was not (Ho et al., 2009b). BAF60c was upregulated ⬃300-fold in
day 10 neurons relative to ES cells (Fig. 5 B, D) in agreement with
previously reported expression in brain and muscle (Wang et al.,
1996b; Lickert et al., 2004). Conversely, BAF60b decreased with
neural differentiation, consistent with previous results that BAF60b
is reduced in cells of the neural lineage (Lessard et al., 2007).
Recently, we found that human fibroblasts can be induced to
neurons with the addition of miR-9/9*and miR-124 and neurogenic factors (Yoo et al., 2011). During the conversion to induced
neurons (iNs), BAF45a was replaced by BAF45b and BAF45c and
BAF53a by BAF53b. We wondered whether SS18 and CREST also
switched with the addition of miR-9/9*-124 and production of
iN. Indeed, CREST was transcriptionally activated ⬎10⫻ in induced neurons, whereas SS18 was transcriptionally reduced 3⫻
(Fig. 6A). The robustness of this switch is such that the level of
CREST expression in the iN is similar to that found in an adult
human brain. Therefore, CREST replaces SS18 during in vivo
neural development, during ex vivo ES-to-neuron differentiation, and also during the conversion of human fibroblasts to
neurons.
SS18 is required for neural stem cell self-renewal
SS18 is a dosage-sensitive gene, being haploinsufficient on the
C57bl6 background with a phenotype of embryonic lethality between E8.5 and E9.5 (De Bruijn, 2006). We were interested in the
role of SS18 in neural stem (NS) cells, but also wondered whether
J. Neurosci., June 19, 2013 • 33(25):10348 –10361 • 10355
there was a difference in the requirement for SS18 in ES cells
compared with NS cells because SS18-null mice develop well past
the peri-implantation stage. Therefore, we hypothesized that NS
cells are more sensitive to reduction in SS18 levels than ES cells.
To test the role of SS18 in ES and NS cell self-renewal, we used
shRNA knock-down delivered by lentiviral vectors to ES cells and
primary neurospheres (NS cells).
Three shRNAs against SS18 were tested, reducing SS18 to
79 ⫾ 4% to 20 ⫾ 1% of the wild-type (WT) protein level. In ES
cells, shSS18 knock-down compared with shScramble control
resulted in a cell-cycle progression phenotype with reduced
S-phase entry and reduced progression from the G2 to the M
phase (Fig. 7A). These reductions in SS18 protein reduced the ES
cell proliferation rates, but were compatible with ES cell culture
viability, because the ES cell cultures continued to proliferate for
at least two passages (Fig. 7B). In NS cells, knock-down of SS18 to
79 ⫾ 4% of WT protein levels resulted in a cell-cycle progression
phenotype with reduced S-phase entry (26.8% WT versus 14.2%
SS18 knock-down) and reduced progression from the G2 to the
M phase (2.4% in G2 for WT vs 8.5% in G2 for SS18 knock-down;
Fig. 7C). A measure of NS cell self-renewal is the ability to form
secondary neurospheres after dissociation to single cells.
shScramble-treated NS cells formed robust secondary neurospheres, but shSS18-treated NS cells failed to form secondary
neurospheres (Fig. 7D). Therefore, in ES cells, SS18 protein could
be reduced 80% and still proliferate, albeit more slowly. However, NS cells were extremely SS18-dosage sensitive, with ⬃25%
reduction causing cell-cycle phenotypes and inhibiting NS cell
self-renewal. We conclude that NS cells are more sensitive than
ES cells to reduced SS18 protein levels, and that this selective
sensitivity is consistent with the pathogenesis of several human
neurologic diseases and syndromes, including microcephaly, resulting from dominant haploinsufficient mutations in npBAF
complexes.
Proper combinatorial assembly of nBAF complexes with
CREST is essential for dendritic outgrowth in neurons
Both BAF53b and CREST have been shown to be required for
activity-dependent dendritic growth (Aizawa et al., 2004; Wu et
al., 2007). We hypothesized that SS18 is transcriptionally repressed at mitotic exit to make way for CREST incorporation into
nBAF complexes, where CREST performs a unique activity that
cannot be replaced by SS18. To test this hypothesis, we forced
SS18 expression in primary cortical neurons and examined
whether dendritic growth was compromised. Depolarization
with KCl induced a robust increase in total dendritic length
in vector and CREST-transfected neurons, whereas SS18transfected neurons had a significant reduction in activitydependent dendritic growth and branch number increase (Fig.
8A–C). Exogenous SS18 and CREST protein incorporated into
BAF complexes (Fig. 8D) and were exclusively nuclear in the
primary cortical neurons (Fig. 8E). Forced expression of SS18
decreased endogenous CREST protein to 50% of control levels in
depolarized cortical neurons, suggesting that exogenous SS18
competes with CREST for incorporation into nBAF complexes
(Fig. 8 D, E). These data indicate that extended expression of SS18
past its normal repression at mitotic exit leads to failure of dendritic outgrowth and that SS18 and CREST are not functionally
redundant as subunits in cell-type specific BAF complexes. The
data also indicate that producing confused nBAF complexes with
SS18 reduces the biological specificity of the nBAF composite
surface and compromises the neuron’s ability to respond productively to depolarization.
10356 • J. Neurosci., June 19, 2013 • 33(25):10348 –10361
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
Figure 7. SS18 knock-down causes a G2-M-phase block and reduced rate of S-phase entry in ES and NS cells and results in failure of NS cells to self-renew. A, ES cell cycle analysis. Bottom left, G1;
top, S-phase (DNA synthesis); bottom right, G2-mitosis. Lentivirus infection with shScramble shSS18#1. shSS18 knock-down causes G2-M-phase block and slower progression into the S-phase. SS18
protein quantification of ES cells. Data are representative of three separate experiments done in duplicate. B, Reduction of SS18 protein with shSS18 slows proliferation rate of ES cells. C, Neurosphere
NS cell-cycle analysis. Infection with shSS18#1 caused a G2-M block and reduced S-phase entry. Shown is SS18 protein quantification of NS cells. The SS18 protein level was reduced ⬃25%, indicating
SS18 haploinsufficiency. Data are shown ⫾ SEM. D, Secondary neurosphere formation is blocked by SS18 knock-down, indicating failure of NS cell self-renewal. Neurosphere NS cell analysis is
representative of two separate experiments done in duplicate. Scale bar, 100 ␮m.
Discussion
The following lines of evidence support our conclusion that SS18
and CREST are stable, dedicated, and functional subunits of the
npBAF and nBAF complexes, respectively: (1) both proteins cosediment exclusively with Brm/Brg on glycerol gradients of
whole-brain preparations, indicating that they are dedicated to
these complexes; (2) they resist dissociation from BAF complexes
even under partially denaturing conditions, indicating that they
make stable, possibly cofolded interactions; (3) they do not co-IP
each other, indicating that they are mutually exclusive subunits;
and (4) SS18 cannot substitute for CREST in supporting dendritic growth. From these studies and their patterns of expres-
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
J. Neurosci., June 19, 2013 • 33(25):10348 –10361 • 10357
Figure 8. SS18 expression prevents KCl-induced dendritic outgrowth. A, Total dendrite length. Primary E18.5 cortical neurons were isolated, transfected with iresGFP, SS18-iresGFP, or
CREST-iresGFP where indicated, cultured for 6 d with 30 mM KCl for the last day where indicated. Control vector and CREST overexpression do not affect dendrite outgrowth in response to KCl
depolarization. SS18 expression reduces activity-dependent dendritic outgrowth in response to KCl depolarization. B, Number of branch points per cell, a measure of dendritic arbor complexity, is
also reduced. The average values are from three independent experiments, with 50 –75 GFP ⫹ neurons scored per condition per experiment. Data are shown ⫾ SEM. *p ⬍ 0.02, **p ⬍ 0.002,
***p ⬍ 0.0005, Student’s t test. C, Representative images of neurons with or without KCl. D, Nuclear extracts of ES cells infected with V5-tagged SS18 and CREST integrate into BAF complexes, as
evidenced by co-IP with Brg and BAF155. E, Exogenous SS18 and CREST protein in cortical neurons is nuclear and SS18 expression in neurons decreases CREST protein levels after depolarization with
KCl. Arrow points to neuron nuclei with decreased CREST. Images are of KCl-depolarized samples. F, Quantification CREST protein level expression (average fluorescence intensity in nuclei). Forced
SS18 expression causes significant decrease in CREST protein with depolarization. The average values are from two independent experiments, with 20 –30 GFP ⫹ neurons scored per condition per
experiment. Dara are shown ⫾ SEM. *p ⬍ 0.02, **p ⬍ 0.002, ***p ⬍ 0.0005, Student’s t test. Scale bar in C, 50 ␮m; in E, 20 ␮m.
sion, we conclude that SS18 is a subunit of esBAF and npBAF
complexes and that CREST is a subunit of nBAF complexes as
illustrated in Fig. 9A. Although we and others have detected SS18
and CREST in studies of BAF complexes, it was not apparent that
they were dedicated, functional, nonexchangable subunits rather
than simply being interacting proteins. From our proteomic
analysis, we conclude that NS/progenitors contain at least 36 different npBAF complexes and that neurons contain at least 36
10358 • J. Neurosci., June 19, 2013 • 33(25):10348 –10361
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
Figure 9. Summary of ordered events in npBAF to nBAF complex switching. A, The npBAF to nBAF switch involves three subunits regulated by miR9/9* and miR-124. Combinatorial assembly of
npBAF and nBAF subunits could create up to 36 distinct npBAF or nBAF complexes. B, In a negative cascade network motif, downstream genes are sequentially repressed and activated when their
regulator reaches the relevant threshold. The conversion of ES cells to neurons illuminates the sequential repression and activation of the genes of the triple-negative miRNA/chromatin switch, which
is a negative cascade network. BAF53b is under independent control for transcription, but the presence of BAF53a will drive BAF53b from the BAF complex and destabilize 53b protein (B.T.S, G.R.C,
unpublished results).
different nBAF complexes. We do not know if these complexes
are present in subtypes of neural progenitors and neurons or if
each neuron has all possible assemblies. From a limited analysis
of different brain regions and inspection of the Allen Brain Atlas
(http://www.brain-map.org/), we favor multiple assemblies in
each neural progenitor or neuron. These findings indicate that
the spectrum of neurologic diseases due to mutations in npBAF
and nBAF complexes must be interpreted in light of the emer-
Staahl et al. • SS18/CREST, Novel Switching Neural BAF Subunits
gent features of different combinatorial assemblies of these
complexes.
SS18 and CREST appear to be functionally exclusive, because
expression of SS18 in postmitotic neurons inhibits activitydependent dendritic outgrowth, whereas expression of CREST
does not. In fact, SS18 expression in neurons phenocopied the
CREST loss-of-function phenotype that is indicative of a
dominant-negative function (Aizawa et al., 2004). We have recently identified two mutations in CREST in amyotrophic lateral
sclerosis (ALS, also known as Lou Gehrig’s disease) patients
(Chesi et al., 2013). We find that these CREST mutants strongly
block dendrite outgrowth even in the presence of endogenous
WT CREST, suggesting a dominant-negative mode of action.
One of the mutations terminates the final 9 aa of CREST, which
has been shown to be necessary for interaction of CREST with
CBP, a histone acetylase (Aizawa et al., 2004). The finding that
CREST is entirely bound to nBAF complexes and resists dissociation even under denaturing conditions indicates that CBP is
targeted to sites of action by nBAF complexes in a way comparable to the targeting of STAT3 to its DNA binding sites by the prior
action of esBAF in ES cells (Ho et al., 2011).
Neither SS18 nor CREST have orthologs in yeast, flies, or
worms. Although BAF complexes are frequently referred to as the
mSWI/SNF complex, only six of the subunits have yeast homologs and other subunits have homologs in the RSC and SWR1
complexes. In most cases, the new BAF subunits appeared either
in insects or fish. An SS18 homolog is present in the zebrafish and
fugu genomes and is present in birds, Xenopus, and mammals
(UCSC Genome Browser; http://www.genome.ucsc.edu/). However, CREST is present in neither the zebrafish nor fugu genomes
but is present in the coelacanth genome. Coelacanths are lobefinned fishes more closely related to tetrapods than to ray-finned
fishes. Therefore, SS18 and CREST appear to have originated to
deal with some aspect of chromatin regulation that has emerged
relatively recently in tetrapod evolution. The exclusive expression
of CREST in the nervous system and the extraordinary sensitivity
of neural progenitors to reduction in SS18 levels indicates that
this recently evolved chromatin function might be used in the
development of the tetrapod motor nervous system. Neither SS18
nor CREST is required for nucleosome remodeling in vitro, indicating they may play a role in genome targeting or some as-yetunrecognized functions of these complexes.
The appearance of CREST in the lobe-finned coelacanth is
interesting because it, like all tetrapods, has limb and finger bones
covered with muscle and skin compared with ray-finned fishes,
which have slender bony fin rays (lepidotrichs) covered with only
skin. One hypothesis is that, during the tetrapod transition,
CREST co-evolved with tetrapod limb development. Indeed, the
majority of the putative regulatory elements located near CREST
appear to originate in the tetrapod ancestor (Lowe et al., 2011).
CREST may play a specific role in motor neuron chromatin regulation, where it pairs with motor neuron factors necessary for
limb neuromuscular development in a way similar to the essential
pairing of BAF60c with myoD to activate the skeletal muscle
program (Albini et al., 2013) or esBAF with Sox2 and Oct4 in ES
cells (Ho et al., 2009a). Perhaps this requirement for CREST in
motor neurons is why mutant CREST causes ALS, a fatal motor
neuron disease characterized by loss of coordination and atrophy
of limbs. Interestingly, CREST-null mice also have coordination
defects and smaller cerebellums (Aizawa et al., 2004). In contrast
to the roles of BAF complexes in mental retardation and microcephaly, ALS is thought to result from deterioration in neural
function and connectivity. Therefore, BAF complexes appear to
J. Neurosci., June 19, 2013 • 33(25):10348 –10361 • 10359
function during normal human neural development and are also
required to maintain circuitry and/or viability over a lifetime.
However, additional studies will be required to confirm these
possibilities.
SS18 and CREST subunits are 62% identical, yet SS18 cannot
substitute for CREST in postmitotic neurons. SS18 has 65 proline
residues and CREST has 27. Proline residues make proteins more
rigid, so the secondary structure of SS18 will be more rigid than
that of CREST and thus could give rise to different functions. In
addition, the paralogs may have different posttranslational modifications, because SS18 has 31 serine and 8 threonine residues
compared with 53 serines and 22 threonines in CREST. There are
two splice variants of SS18, the short isoform (lacking exon 8)
being the more abundant of the two (Fig. 5B). Both SS18 splice
variants functioned as a dominant-negative and blocked activitydependent dendritic outgrowth (Fig. 8 and data not shown). This
indicates that exon 8, which CREST contains, does not confer
CREST-like function to SS18. Therefore, the unique functionality of SS18 and CREST must come from other biochemical
differences.
Recent exome sequencing studies have revealed frequent mutations in BAF complex subunits in CSS, NBS, and sporadic mental retardation. In addition, mutations have been reported in Brg
and Brm in schizophrenia and in BAF155, BAF170, BAF180, and
REST in human autism (Neale et al., 2012; O’Roak et al., 2012).
These mutations were dominant and considered to be dominantnegatives. In the NBS study, the authors suggested the dominant
mutations in BRM (one of two ATPases that in neurons are components of neural-specific nBAF complexes) to be dominantnegatives and also affect Brg-based complexes. Genetic
dominance can have many different mechanistic underpinnings,
including the production of dominant-negative proteins that can
block WT protein function, abnormal polymerization processes
in which the mutated protein blocks polymer extension, and haploinsufficiency due to a nonsense mutation in a protein involved
in a rate-limiting process. To clarify this issue, we reexamined our
proteomic analysis done with purified nBAF complexes. We
found that peptides from Brg exceeded peptides recovered from
Brm by more than threefold. Because both are large proteins,
these differences are unlikely to be due to peptide recovery differences and likely reflect the endogenous levels of the proteins.
Many of these BRM dominant mutations are surprisingly conservative amino acid changes and cluster to the ATP binding and
Helicase domains (Ronan et al., 2013), suggesting that rather
than being strong dominant-negatives, the mutations affect a
rate-limiting catalytic process. ATP-dependent chromatin remodeling is well suited for a rate-limiting role in biological processes, because the rate of chromatin alteration may be limited by
the intrinsic rate of energy utilization by these complexes rather
than by ATP levels, which are generally not rate limiting. The
dominant role of BAF subunits in human diseases fits nicely with
the observation that, in mice, mutations in Brg, BAF155, and
BAF53a all have dominant phenotypes and are apparently haploinsufficient. Rate-limiting functions are often instructive, so
these considerations are consistent with the apparent instructive
role of the microRNA/chromatin switch in converting fibroblasts
to neurons (Yoo et al., 2011). Interestingly, the ALS mutations we
have described also appear to be dominant (Chesi et al., 2013).
Our kinetic analysis of conversion of ES cells to neurons reveals the nature of the negative cascade network motif underlying
the triple-negative genetic circuit we described as contributing to
neural fate determination (Yoo et al., 2009; Yoo et al., 2011). The
timing of repression of REST, activation of mir-9/9* and miR-
10360 • J. Neurosci., June 19, 2013 • 33(25):10348 –10361
124, subsequent repression of BAF53a, and stabilization of
BAF53b protein nicely fit the negative cascade network motif
model in which downstream genes are sequentially repressed and
activated when their regulators reach the relevant threshold
(Shoval and Alon, 2010; Fig. 9B). Our kinetic analysis also indicates that more than a single pathway controls the microRNA/
chromatin switch. This follows from the observation that BAF45c
is induced without RA and before miR-9/9* and miR-124. The
requirement for two pathways to bring about this epigenetic
switch indicates that the npBAF to nBAF switch could be a hub or
integration site for neurogenesis, which is supported by recent
genome sequencing efforts in human neurologic diseases.
The discovery that CREST is a dedicated, nonexchangeable
subunit of nBAF complexes indicates that these complexes have
three neuronal-specific “letters” endowing them with the ability
to provide specific patterns of gene expression in neurons. These
complexes in neurons are additionally polymorphic by virtue of
alternative assembly of other subunits such as Brg versus Brm,
BAF250a versus BAF250b versus BAF200, BAF60a versus
BAF60b versus BAF60c, and BAF45b versus BAF45c. Therefore,
they should be referred to as npBAF complexes and nBAF complexes, each with a complexity of 36 possible assemblies based on
gene numbers (Fig. 9A). An even larger number of combinatorial
possibilities might result from the extensive alternative splicing
shown for BAF250b and SS18. The functional target(s) of the
neuron-specific surfaces of the nBAF complexes is not known,
but is likely to reflect the emergent features of complexes (i.e.,
those that emerge not from the subunit itself, but rather from the
composite surface produced by each subunit and its adjacent
subunits). Our findings indicate that the widely different neurologic diseases causally or possibly related to mutations in BAF
subunits (mental retardation, schizophrenia, autism, ALS, and
microcephaly) must be interpreted in the context of these different BAF assemblies rather than a monomorphic mSWI/SNF
complex.
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